The long-range goal of our research program is to understand the molecular events underlying function of the heterodimeric sweet taste receptor (T1R2+T1R3). The sweet receptor is a remarkably broadly acting receptor, capable of responding to native and artificial sweeteners. In vivo and in vitro studies suggest that this single heterodimeric receptor is the primary or only sweet taste receptor. We are interested in how so many chemically diverse ligands can bind to the sweet receptor, how binding at different sites leads to receptor activation, and how the domains of each T1R monomer contribute to binding, activation and signal transduction. To address these goals we have developed ligand binding and activity assays, and used these techniques in concert with mutagenesis and molecular modeling to begin to understand this complex receptor. The present proposal applies these several techniques to examine how the small molecule-binding site of T1R2 interacts with aspartame, neotame and alitame (so-called dipeptide sweeteners). This "canonical" binding site is found within the "venus fly trap module" (VFTM) of T1R2. In Aim 1 we will use the differential sensitivity of the human and mouse sweet receptors to dipeptide sweeteners, along with heterologous expression assays, to identify key residues within the VFTM of T1R2 involved in the interactions with dipeptide sweeteners. In Aim 2 we will use directed mutagenesis of T1R2, heterologous assays and molecular modeling to physically and chemically characterize the interaction of dipeptide sweeteners with the VFTM of T1R2. In Aim 3 we will use spectroscopic and calorimetric techniques to monitor binding of dipeptide sweeteners to the expressed VFTM of T1R2 and T1R2 mutants. There is today in the affluent countries of the world an epidemic of obesity, insulin-resistant diabetes and diet-related disorders. In our evolutionary past a strong drive to consume high-carbohydrate/energy-rich foods was advantageous for survival. Today, our more sedentary lives and the ready availability of food makes this sweet-seeking behavior a liability that may contribute significantly to obesity. Sweet taste perception mediated by the heterodimeric sweet taste receptor undoubtedly contributes to sweet-seeking behavior and food consumption. The studies in this proposal will enhance our understanding at the molecular level of sweet receptor function with the hope of future means to control our sweet cravings and the attendant diseases of over-consumption. [unreadable] [unreadable] [unreadable]